Complete assignments of NMR data and assessment of trypanocidal activity of new eremantholide C derivatives

Abstracts

Chemical transformations of eremantholide C (1), a sesquiterpene lactone that was isolated from Lychnophora trichocarpha Spreng. led to five new derivatives: 1′,2′- epoxyeremantholide C (2), 5-n-propylamine-4,5-dihydro-1′,2′-epoxyeremantholide C (3), 5-n-propylammonium-4,5-dihydro-1′,2′-epoxyeremantholide C chloride (4), 5-n-propylammonium-4,5-dihydroeremantolide C chloride (5) and 16-O-ethyleremantholide C (6). The structures of all these derivatives were assigned on the basis of IR, MS, ¹H and ¹³C NMR data by 1D and 2D techniques. Eremantholide C and the derivatives 2, 4 and 5 were evaluated against trypomastigotes Y and CL strains of Trypanosoma cruzi. Eremantholide C completely inhibited the growth of both the parasites strains while all derivatives were partially active against the CL strain and inactive against the Y strain.

Eremantholide C derivatives; Lychnophora trichocarpha ; NMR; sesquiterpene lactones; trypanocidal activity

Transformações químicas realizadas em eremantolida C (1), uma lactona sesquiterpênica isolada de Lychnophora trichocarpha Spreng. originaram cinco novos derivados: 1′,2′-epoxieremantolida C (2), 5-n-propilamino-4,5-diidro-1′,2′-epoxieremantolida C (3), cloreto de 5-n-propilamônio-4,5-diidro-1′, 2′-epoxieremantolida C (4), cloreto de 5-n-propilamônio-4,5-diidroeremantolida C (5) e 16-O-etileremantolida C (6). As estruturas químicas de todos estes derivados foram elucidadas com base nos espectros de IV, EM, RMN de ¹H e de ¹³C e por meio de técnicas 1D e 2D. Eremantolida C e os derivados 2, 4 e 5 foram avaliados frente a cepas tripomastigotas Y e CL de Trypanosoma cruzi. Eremantolida C inibiu completamente o crescimento de ambas as cepas de parasitas, enquanto todos os derivados foram parcialmente ativos contra a cepa CL e inativos contra a cepa Y.

Derivados de eremantolida C; Lychnophora trichocarpha ; RMN; lactonas sesquiter-pênicas; atividade anti-Trypanosoma curuzi

INTRODUCTION

Sesquiterpene lactones are chemical markers of certain plant families such as Asteraceae. The large number of biological activities experimentally described up to now, raised great interest on this group of substances. The sesquiterpene lactones of the furanheliangolide type are biogenetically derived from heliangolides and are often found in species of the genus Lychnophora, that is native from Brazil (Bohlmann and Jakupovic 1990). In previous studies, eremantholide C and its oxidized derivatives showed activity against Trypanosoma cruzi (Oliveira et al. 1996, Saúde-Guimarães et al. 2007). Other biological/pharmacological activities were reported for eremantholide C, such as antibacterial, anti-hyperuricemic, anti-gouty arthritis, anti-inflammatory and antitumor (Barrero et al. 2000, Saúde et al. 2002, de Souza et al. 2012, Ferrari et al. 2013, Saúde-Guimarães et al. 2014). Aiming to obtain new bioactive derivatives of eremantholide C (Figure 1), this sesquiterpene lactone was submitted to chemical modifications.

Figure 1
Eremantholide C and derivatives 2-6.

MATERIALS AND METHODS

General Procedures

Mass spectra were obtained with VG Autospec (electron impact) and HP 5988A (chemical ionization with methane) spectrometers by direct injection (ionization chamber at 200 °C). IR spectra were taken at Galaxy 3000-FTIR spectrophotometer (Mattson Instruments). NMR spectra were taken at Bruker Avance DPX (4.7T), DRX (9.4T), equipped with a 5mm dual probe, at 300 K, with TMS as internal reference. One-dimensional ¹H and ¹³C NMR spectra were acquired under standard conditions, with 90° pulse widths of 8.00 µs and 8.50 µs for ¹H and ¹³C, respectively. ¹H NMR spectra were obtained using a sweep width of 3 kHz over 32k data points. ¹³C NMR spectra were obtained using a sweep width of 31 kHz. DEPT, ¹H, ¹H COSY and ¹H, ¹³C HETCOR techniques were performed using standard pulse sequences supplied by the spectrometer manufacturer.

Synthesis of Eremantholide C derivatives 2 to 6

Eremantholide C (1) was obtained from aerial parts of L. trichocarpha Spreng. that were collected in Minas Gerais, Brazil, as previously described (Saúde et al. 1998, Ferrari et al. 2013). Its structural characterization is described elsewhere (Le Quesne et al. 1978, Saúde et al. 1998, Saúde-Guimarães et al. 2007).

1′,2′-Epoxyeremantholide C (2) was prepared by reaction of 1 (2.9 mmol) with m-chloroperbenzoic acid (5.3 mmol) in 50 mL of anhydrous CHCl₃. The mixture was stirred at room temperature for 2 h. The reaction mixture was worked out according to Carda et al. 1986. The resulting residue (1.14 g) was purified by column chromatography (silica gel, hexane: EtOAc 1:1), yielding 970 mg of 2 (93% yield, m.p. 213-217 °C).

1′,2′-Epoxyeremantholide C (2):6,9-Epoxy-2H-1,4-dioxacyclodeca[c,d]pentalene-2,7(4aH)-dioxane,2a,3,5,6,11a,11b-hexahydro-3-hydroxy-2a,6,10-trimethyl-3-(1′,2′-epoxy)-2aR*,3S*,4a R*,6S*,10Z,11aS*,11bS*). White solid, m.p. 213-217°C. IR (KBr) ν _max(cm^–1): 3400 (OH), 1770 (C=O, γ-lactone), 1700 (ketone C=O), 1650 (C=C), 1590 (C=COR, furanone), 1450, 1350, 1300, 1275, 1150, 1100, 1050, 1000, 900. MS (EI), m/z(rel. int.): 362 [M⁺, C₁₉H₂₂O₇] (20), 344 (M – H₂O, 17), 234 (19), 205 (19), 189 (20), 177 (29), 165 (32), 149 (25), 138 (55), 122 (53), 95 (100), 69 (58), 57 (22).

5-n- Propylamine -4,5-dihydro-1′,2′-epoxye-remantholide C (3) was obtained by reaction of 2(0.55 mmol) with n-propylamine (5.94 mmol), at -18 °C for 16 h (Kirk 1973). After this period, the excess of the amine was removed by evaporation under reduced pressure, at room temperature. The residue obtained was purified by PTLC on silica gel (0.5 mm thickness, eluent - hexane: EtOAc 1:1) resulting in 82 mg (35% yield) of 3 as a white solid. This compound was dissolved in dry THF and treated with gaseous HCl leading to the formation of 4 as a white hydrosoluble solid (quantitative yield).

5-n-Propylamine-4,5-dihydro-1′,2′- epoxyeremantholide C (3): White solid, m.p. 183-189 °C. IR (KBr) ν_max(cm^–1): 3400 (OH), 1770 (C=O, γ-lactone), 1700 (C=O, ketone), 1590 (C=COR, furanone), 1450, 1380, 1300, 1200, 1150, 1100, 1050, 1000, 900, 850. MS (EI), m/z (rel. int.): 421 (M⁺, C₂₂H₃₁O₇N, 99), 403 (M - H₂O, 28), 238 (25), 195 (64), 168 (31), 151 (25), 138 (33), 125 (100), 99 (23), 72 (35).

5-n-Propylammonium-4,5-dihydro-1′,2′-epoxyeremantholide C chloride (4): white solid, m.p. 176-179 °C. MS (EI), m/z (rel. int.): 457 (M⁺, C₂₂H₃₂O₇NCl, 72), 439 (M - H₂O, 11), 390 (18), 364 (31), 336 (21), 318 (59), 195 (79), 168 (44), 152 (19), 138 (45), 125 (100), 99 (45), 81 (18), 72 (46).

5-n-Propylammonium-4,5-dihydroereman-tolide C chloride (5) was obtained from the reaction of 1(0.58 mmol) with n-propylamine (5.94 mmol), at -18 °C for 10 h (Kirk 1973). After this period, the excess of amine was removed by evaporation under reduced pressure, at room temperature. The residue obtained was purified by PTLC on silica gel (0.5 mm thickness, eluent - hexane: EtOAc 1:1) affording 120 mg of a yellowish pasty material. Silica TLC of this material showed three spots when revealed by iodine. After development with ninhydrin, the TLC showed only a rosy spot, characteristic of amines. This material was then dissolved in dry THF and treated with gaseous HCl to give the hydrochloride 5 as a white hydrosoluble solid (70 mg).

5-n-Propylammonium-4,5-dihydroere-mantolide C chloride (5): White solid, m.p. 195-197 °C. MS (EI), m/z (rel. int.): 405 (60), 387 (M - H₂O, 5), 195 (40), 168 (32), 135 (21), 125 (100), 69 (35).

16-O-Ethyleremantholide C (6) was obtained, according to the methodology described by Partwardhan et al. 1974, by reacting 1 (0.28 mmol) with 0.08 mL of triethyl orthoformate and 25 mg of Amberlyst resin 15. The reaction mixture was stirred at room temperature for 3 days. Then it was neutralized with aqueous K₂CO₃ solution and filtered. The product was extracted with diethyl ether. The organic layer was washed with water, filtered on anhydrous sodium sulfate and concentrated under reduced pressure, yielding 72 mg of ether 6 as a white solid (66% yield).

16-O-Ethyleremantholide C (6): White solid. IR (KBr) ν_max(cm^–1): 1770 (C=O, γ-lactone), 1700 (C=O, ketone), 1660 (C=C), 1590 (C=COR, furanone), 1450, 1380, 1300, 1280, 1260, 1210, 1200, 1150, 1140, 1100, 1060, 1000, 870, 750. MS (EI), m/z (rel. int.): 375 (M+1)⁺, C₂₁H₂₆O₆, 3), 329 (54), 285 (17), 199 (9), 165 (49), 95 (85), 69 (100), 67 (18), 45 (23), 43 (39).

In vitro Assays with Trypanosoma cruziTrypomastigotes

Albino mice infected with the Y or CL strains of T. cruzi were used to provide trypomastigotes.

Samples of eremantholide C and of the derivatives 2, 4 and 6 were dissolved or suspended in dimethyl sulfoxide (DMSO) (0.2 mL) and plus Krebs-Ringer-glucose (2.0 mL) and mixed with an equal volume of parasitized whole blood diluted in bovine calf serum. A parasite density of 2 x 10⁵trypomastigotes/0.1 mL was calculated for each flat-bottomed test tube (4 mL, 56 x 13 mm); control tubes without the test extracts were included. After incubation at 4 °C for 24 h the suspensions were examined microscopically. Only those samples that killed 100% of the parasites were considered active. Samples that inhibited 50% of parasite growth compared to control were considered partially active.

RESULTS AND DISCUSSION

Data of the ¹H and ¹³C NMR spectra of derivatives 2-6 are given at on Tables I and II, respectively. The chemical shifts were assigned by consideration of known substituent effects of the groups concerned and with the aid of both ¹H,¹H COSY and ¹H,¹³C HETCOR contour maps. The stereochemistry of the carbons were determined based on coupling constants and ¹H,¹H NOESY contour maps.

TABLE I
¹H NMR data for compounds 1, 2, 3, 4, 5 and 6, δ, J (Hz).

H	1*,1	2**,1	3**,1	4**,2	5**,2	6**,1
2	5.63 s	5.62 s	5.56 s	5.99 s	5.93 s	5.62 s
4	-	-	2.89 q J 4,15 = 7.2	3.56 q J4,15 = 7.2	3.45 q J 4,15 = 7.2	-
5	6.04-6.03 m	6.03-6.00 m	2.99 brs	3.97 brs	3.88 brs	6.03-6.01 m
6	5.02-4.98 m	5.02-4.97 m	4.26 d J6,7 = 6.5	4.74 a	4.73 d J6,7 = 7.9	4.90-4.86 m
7	2.82 dd J6,7 = 7.1; J7,8 = 4.2	2.87 dd J6,7 = 7.3; J7,8 = 4.2	2.95 dd J6,7 = 6.5; J7,8 = 4.9	3.09 dd J6,7 = 7.8; J7,8 = 4.2	3.04 dd J6,7 = 7.8; J7,8 = 4.3	2.84 dd J6,7 = 7.2; J7,8 = 4.4
8	4.09 ddd J7,8 = 4.2; J8,9a = 2.5; J8,9b = 12.0	4.07 ddd J7,8 = 4.2; J8,9a = 2.6; J8,9b = 11.9	3.91 ddd J7,8 = 4.9; J8,9a = 2.3; J8,9b = 11.9	3.92 m	3.96 ddd J7,8 = 4.3; J8,9a = 2.8; J8,9b = 113	3.85 ddd J7,8 = 4.4; J8,9a = 2.4; J8,9b = 12.0
9a	2.48 dd J8,9a = 2.5; J9a,9b = 13.5	2.39 dd J8,9a = 2.6; J9a,9b = 13.6	2.39 dd J8,9a = 2.3; J9a,9b = 13.5	2.32 dd J8,9a = 2.3; J9a,9b = 13.6	2.27 dd J8,9a = 2.8; J9a,9b = 13.8	2.41 dd J8,9a = 2.4; J9a,9b = 13.6
9b	2.00 dd J8,9b = 12.0; J9a,9b = 13.5	1.94 dd J8,9b = 11.9; J9a,9b = 13.6	1.90 dd J8,9b = 11.9; J9a,9b = 13.5	2.39 J8,9b = 11.5; J9a,9b = 13.6	2.34 dd J8,9b = 11.3; J9a,9b = 13.8	2.06 dd J8,9b = 12.0; J9a,9b = 13.6
13	1.18 s	1.33 s	1.38 s	1.39 s	1.21 s	1.16 s
14	1.45 s	1.48 s	1.43 s	1.50 s	1.45 s	1.50 s
15	2.05 t J5.15 =6,15= 1.9	2.05 t J5,15 =6,15= 2.0	1.37 d J4,15 = 7.2	1.52 d J4,15 = 7.2	1.48 d J4,15 = 7.2	2.06 dd J5,15 = 2.0; J6,15 = 1.6
2′a	5.31 brs	3.11 d J2′a,2′b = 5.5	3.12 d J2′a,2′b = 5.5	4.04 d J2′a,2′b = 11.9	5.22 brs	5.28 d J2′a,2′b = 1.2
2′b	5.07 t J2′a,2′b = 1.6	2.70 d J2′a,2′b = 5.5	2.71 d J2′a,2′b = 5.5	3.75 d J2′a,2′b = 11.9	5.13 t J2′a,2′b = 1.4	5.13 brs
3′	1.91 s	1.55 s	1.57 s	1.50 s	1.77 s	1.79 s
4′a	-	-	2.8 dt J4′a,4′b = 11.2; J4′a,5′ = 7.0	3.28 td J4′a,4′b = 11.2; J4′a,5′ = 5.8;	3.18 dt J4′a,4′b = 10.5; J4′a,5′ = 5.7	3.36 dq J4′a,4′b = 9.2; J4′a,5′ =7.2
4′b	-	-	2.53 dt J4′a,4′b = 11.2; J4′b,5′ = 6.9;	3.17 td J4′a,4′b = 11.2; J4′b,5′ =5.8	3.09 dt J4′a,4′b = 10.5; J4′b,5′ =5.7	3.20 m
5′	-	-	1.50 m	1.80 m	1.75 m	1.07 t J4′a,5′ =,4′b,5′ = 7.2
6′	-	-	0.93 t J5′,6′ = 7.3	0.99 t J5′,6′ = 7.4	0.94 t J5′,6′ = 7.3	-
OH	3.79 s	3.69 s

• x – Superimposed signal with D₂O.
• ^*
  - 300 MHz;
• ^**
  - 400 MHz,
• 1 - CDCl₃, 2 - D₂O.

TABLE II
¹³C NMR data (δ) for 1, 2, 3, 4, 5 and 6.

C	1*,1	2**,1	3**,1	4**,2	5**,2	6**,1
C-1	205.89	205.23	204.53	209.06	209.34	205.24
C-2	104.54	104.50	104.03	105.93	106.08	104.45
C-3	187.27	186.64	192.93	192.39	192.53	186.91
C-4	130.00	130.15	38.14	33.44	33.68	139.10
C-5	134.77	134.28	57.18	58.65	57.30	134.75
C-6	81.46	81.34	82.42	80.77	81.00	81.75
C-7	62.53	63.06	65.24	63.79	63.99	63.10
C-8	78.37	78.38	77.45	76.47	77.05	78.43
C-9	43.46	43.25	43.84	42.38	42.60	43.89
C-10	90.24	89.95	89.88	91.88	91.99	89.98
C-11	59.88	58.43	58.51	61.65	60.61	60.00
C-12	175.72	174.70	175.09	177.21	178.19	175.76
C-13	21.94	20.64	21.16	21.52	20.83	21.66
C-14	20.48	20.39	20.51	20.16	20.22	20.64
C-15	20.30	20.33	16.70	15.06	15.17	20.49
C-16	106.09	104.57	104.94	107.06	107.27	109.59
C-1′	142.22	59.60	59.71	76.67	141.39	139.28
C-2′	115.80	53.59	53.80	50.06	116.63	117.61
C-3′	19.00	11.44	17.55	20.07	19.19	18.46
C-4′	-	-	50.51	49.11	49.22	57.26
C-5′	-	-	17.52	19.04	18.70	15.29
C-6′	-	-	11.65	10.52	10.62	-

• ^*
  - 75 MHz;
• ^**
  - 100 MHz;
• 1 - CDCl₃; 2 - D₂O.

From the reaction of 1 with m-chloroperbenzoic acid, the main product 2 was obtained and it was characterized by ¹H NMR data. The presence of an epoxy group in 2 was highlighted in the ¹H NMR spectrum by the two doublets at δ3.11 (J = 5.5 Hz) and δ2.70 (J = 5.5 Hz), attributed to H-2′a and H-2′b, which appear in the ¹H NMR spectrum of 1 at δ5.31 (br s) and δ5.07 (t, J = 1.6 Hz), respectively.

The signals at δ130.00 and δ115.80, attributed to the olefinic carbons C-1′ and C-2′, respectively, in the ¹³C NMR spectrum of 1, are replaced in the spectrum of 2 by the signals at δ59.50 and δ58.47, typical chemical shifts of oxygenated carbons with hybridization sp³. In addition, the signal attributed to C-3 in the spectrum of 1 (δ19.00) is shifted to δ11.44 in the spectrum of 2, confirming the epoxidation of the Δ¹ double bond of 1.

The mass spectrum of 2 showed the molecular ion peak at m/z 362, which represents an increment of 16 mass units to the molar mass of 1, corresponding to the addition of an oxygen atom. This mass is consistent with the molecular formula C₁₉H₂₂O₇.

Data of IR, MS, ¹H NMR and ¹³C NMR of 2, are in agreement with the new derivative 1′,2′-epoxyeremantholide C.

Epoxide 2 was reacted with methylamine, cyclohexylamine, diethylamine and n-propylamine. Reactions with the first three amines led to mixtures of many products, in such a way that no derivative with suitable purity was obtained. The reaction of 2 with n- propylamine afforded a less complex mixture of products which, after separation by column chromatography on silica gel, led to the amino product 3 with a 35% yield. The amine 3 was transformed into its hydrochloride salt by reaction with gaseous HCl in dry THF, leading to the hydrosoluble compound 4.

The multiplets in δ5.02-4.98 and δ6.03-6.00 attributed, respectively, to H-6 and H-5, in the ¹H NMR spectrum of 2appear in the spectrum of 3 at δ4.26 (d, J = 6.5 Hz) and at δ2.99 (brs), respectively. The quartet at δ2.89 (J = 7.2 Hz), that appears in the ¹H NMR spectrum of 2, was assigned to H-4. A correlation of this signal with the doublets at δ1.37 (J = 7.2 Hz), assigned to H-15, is indicated by the values of the coupling constants and by the COSY ¹H-¹H contour map. The signals at δ134.27 and δ130.24 which are attributed to the olefinic carbons C-5 and C-4, respectively, as well as the signal attributed to C-15 (δ20.39) in the ¹³C NMR spectrum of 2, are shifted in the ¹³C NMR spectrum of 3 to δ65.24, δ38.20 and δ16.70, respectively, allowing the proposition that the amine group was linked to the C-5 of compound 2.

The double triplets at δ2.80 (J = 7.0 and 11.2 Hz) and δ2.53 (6.9 and 11.2 Hz) assigned to H-4′a and b, respectively, the multiplet at δ1.50 assigned to H-5′, and the triplet at δ0.93 (J = 7.3 Hz) assigned to the atoms H-6′ of the methyl group, indicated the presence of a propylamine group at 3. This group was confirmed by the ¹³C NMR signals at δ50.51, δ17.52 and δ11.65, attributed to carbons 4′, 5′ and 6′, respectively.

The two doublets at δ2.71 (J = 5.5 Hz) and δ3.12 (J = 5.5 Hz) assigned to H-2′a and b, respectively, and the signals at δ59.71 and δ53.80 on the ¹³C NMR spectrum, due to carbons 1′ and 2′, respectively, showed that the epoxy group remained unchanged in the molecule of 3.

The assignments made to the signals in the ¹H NMR spectrum of 3 were confirmed by the values of the coupling constants and by the ¹H-¹H COSY contour map which showed the following correlations: H-4 and H-15; H-4′a, H-4′b and H-5′; H-5′ and H-6′; H-6 and H-7, and H-2′a and b. Chemical shifts for ¹³C NMR spectrum of 3 were assigned by comparison with the spectrum of 2 and based on the DEPT spectrum.

The mass spectrum of 3 presented the molecular ion peak at m/z 421 u, corresponding to 59 mass units higher than the molecular ion of 2, and it corresponds to n-propylamine group (C₃H₇NH₂). The molar mass of 3 was compatible with the molecular formula C₂₂H₃₁O₇N.

Based on data retrieved from IR, MS, ¹H and ¹³C NMR it was concluded that the product obtained was that resulting from the amine addition to carbon-5 of the epoxide 2, through a Michael's reaction, generating the novel derivative 5-n-propylamine-1′,2′-epoxyeremantholide (3). This might be compared to the proposed mechanism for the antitumor activity of the eremantholides by reaction with biological nucleophiles (Mc Dougal et al. 1989).

The shifting of the signals due to the hydrogen and carbon atoms 4, 5, 15, 4′a, 4′b, 5′ e 6′ of compounds 3 and 4 could be observed by comparing their ¹H and ¹³C NMR spectra.

The signals relative to H-4, H-5, H-15, H-4′a and H-4′b, H-5′ and H-6′ appear in the spectrum of 3, respectively, at δ2.89 (q, J = 7.2 Hz), δ2.99 (brs), δ1.37 (d, J = 7.2 Hz), δ2.80 (dt, J = 7.0 and 11.2 Hz), δ2.53 (dt, J = 6.9 and 11.2 Hz), δ1.50 (m) and δ0.93 (t, J = 7.3 Hz), while in the spectrum of 4 they are shown at δ3.56 (q, J = 7.2 Hz), δ3.97 (brs), δ1.52 (d J = 7.2 Hz), δ3.28 (td, J = 5.8 and 11.2 Hz), δ3.17 (td, J = 5.8 and 11.2 Hz), δ1.80 (m) and δ0.99 (t, J = 7.4 Hz), respectively.

In the ¹³C NMR spectrum of 3, the signals at δ 38.20, δ 57.18, δ 16.70, δ 50.51, δ17.52 and δ11.65 were assigned to C-4, C-5, C-15, C-4′, C-5′ and C-6′, respectively. These signals were observed at δ33.44, δ58.65, δ15.06, δ49.11, δ19.04 and δ10.52, respectively, in the spectrum of 4.

The signal corresponding to H-6 in the ¹H NMR spectra of 4 overlapped that of the solvent (D₂O) at δ4.74. This was confirmed by the ¹H-¹H COSY contour map of 4, evidencing correlations between the signal at δ4.74 (brs), and the signals attributed to H-7 (δ3.09, dd, J = 4.2 and 7.8 Hz), and to H-5 (δ3.97, brs). The ¹H-¹H COSY also evidenced correlations between the following hydrogen atoms: H-4 and H-15; H-8, H-9a and H-9b; H-9a and H-9b; H-4′a, H-4′b and H-5′; H-5′ and H-6′; H-2′a, H-2′b and H-3′.

The relative configuration of the n-propylamine and the C-15-methyl group was defined on the basis of the ¹H,¹H NOESY (Figure 2), that indicated correlation between the H-5 signal at δ3.97 (brs), with the signals at δ4.74 and δ3.56 (q, J = 7.2 Hz), attributed to H-6 and H-4, respectively. This correlation showed a –cisrelationship between the H-4 and H-5 atoms with a β configuration. Consequently, the C-15-methyl group and the n-propylamine group should have an α configuration.

Figure 2
Arrows represent NOESY correlations.

The mass spectrum of 4 presented a molecular ion peak m/z 457 u, compatible with a molecular formula C₂₂H₃₂O₇NCl. Such a molecular mass represents an increment of 36 units compared with the mass of 3, compatible with the addition of one HCl per molecule of 4.

Based on data from IR, MS, ¹H and ¹³C NMR, COSY and NOESY it was concluded that compound 4 is 5-n-propylammonium-4,5-dihydro-1′,2′-epoxyeremantholide C chloride, a novel compound that is firstly described in this paper.

In order to confirm the positioning of the n-propylamine group at the C-5 of compound 2, the reaction of 1 with n-propylamine was carried out. Three spots were observed when a silica gel TLC plate of the reaction mixture was sprayed with iodine. Under ninhydrin, only a rosy spot typical of amine, was observed. Then, the mixture was treated with gaseous HCl in dry THF yielding 5 as a white hydrosoluble solid.

In the ¹H NMR spectrum of 5, the signals at δ3.18 (td, J = 5.7 and 10.5 Hz), δ3.09 (td, J = 5.7 and 10.5 Hz), δ1.75 (m) and δ0.94 (t, J = 7.3 Hz) were attributed to H-4′a, H-4′b, H-5′ and H-6′ of the n-propylammonium group, respectively. These data were further confirmed by the signals at δ49.22 and δ18.70, together with the signal at δ10.62 in the ¹³C NMR of 5, which were attributed, respectively, to C-4′, C-5′ and C-6′ of the n-propylammonium group.

The ¹H NMR spectrum of 5 presented a quartet at δ3.45 (1H, J = 7.2 Hz) that was attributed to H-4. The signals for H-5 and H-15 in this spectrum appeared as a broad singlet at δ3.88 and a doublet at δ1.48 (J=7.2 Hz), respectively. In the ¹H NMR spectrum of 1 these atoms are represented by signals at δ6.04–6.03 (m) and δ2.05 (t, J = 1.9 Hz), respectively. Values of coupling constants for H-4 and H-15 indicate that these hydrogens are in vicinal positions.

The signal attributed to H-6 in the ¹H NMR spectra of 5 is probably superimposed to that of H₂O in the solvent (D₂O), which appear at δ4.74. In order to shift the signal of H₂O and observe the one from H-6, the ¹H NMR spectra of 5 in D₂O was run at 330 K, when the signal due to the solvent was shifted to δ4.72 and the doublet attributed to H-6 was seen at δ5.07 (J = 7.9 Hz).

The stereochemistry of the n-propylammonium and of the C-15 methyl groups were inferred based on the NOESY technique (Figure 2), which indicated a correlation between the signal at δ3.88 (brs), attributed to H-5, and the signals at δ4.73 (d, J = 7.9 Hz) and δ 3.45 (q, J = 7.2 Hz), corresponding to H-6 and H-4, respectively. These data suggest that H-4 and H-5 have a cis relationship and a β configuration. Consequently, the C-15 methyl and the n- propylammonium groups have a α configuration.

Themolecularionpeakm/z441 u, corresponding to the molecular formula C₂₂H₃₂O₆NCl, was not observed at the mass spectrum of 5, but it showed a peak at m/z 405 u that was compatible to the loss of a molecule of HCl from the molecular ion.

Data of IR, MS, ¹H and ¹³C NMR spectra, and the COSY and NOESY contour maps are in agreement with the new derivative 5-n- propylammonium-4,5-dihydroeremantholide C chloride, a new derivative of eremantholide C.

The infrared spectrum of 6 showed no absorption characteristic of the hydroxyl group. The ¹H NMR spectrum presented signals at δ3.36 (dq, J = 7.2 and 9.2 Hz), δ3.20 (m) and δ1.07 (t, J = 7.2 Hz) corresponding to H-4′a, H-4′b and H-5′, respectively, and that indicates the presence of an ethoxyl group. The COSY ¹H,¹H contour map evidenced correlations between H-4′a, H-4′b and H-5′ atoms.

The ¹³C NMR spectrum of 6 shows a signal at δ57.26, characteristic for a sp³ carbon bonded to oxygen and, therefore, it was attributed to C-4′. The signal at δ15.29 was assigned to the methyl carbon at the 5′ position. These data confirmed the presence of an ethoxyl group in the molecule of 6.

In the mass spectrum of 6 the ion [M+1]⁺ appeared at m/z 375 u corresponding to an increase of 29 mass units in comparison with 1, that is compatible with the presence of an ethyl group, and corresponding to the molecular formula C₂₁H₂₆O₆.

Assignments of the signals in the ¹H NMR spectrum of 6 were confirmed through the correlations observed at the ¹H, ¹H COSY contour map.

Based on the modifications observed in the IR, MS, ¹H and ¹³C NMR spectra it was concluded that the compound obtained was the new derivative 16-O-ethyleremantholide C (6).

Eremantholide C derivatives 2, 4 and 5 were tested against trypomastigote forms of Y and CL strains of Trypanosoma cruzi, the infectious agent of Chagas' disease, in comparison with Violet crystal (active at 125 µg.mL^–1) that was used as reference in the in vitro tests. The results are shown on Table III. Eremantholide C and the derivatives 2, 4 and 5 were evaluated against Y and CL strains of T. cruzi. Eremantholide C completely inhibited the growth of both the parasite strains in the concentrations of 3,600 µg/mL (Y strain) and 1,800 µg/mL (CL strain), respectively, while all the derivatives were partially active against the CL strain and inactive against the Y strain, in the concentrations assayed.

TABLE III
Results of in vitro tests of compounds 1, 2, 4 and 5 against Y and CL trypomastigote strains of T. cruzi. Inhibition of growth (%).

Compound	Y Strain (µg/mL)	CL Strain (µg/mL)
1	3600 (100%)	1800 (100%)
2	3290 (NI)	823 (50%)
4	4155 (NI)	1039 (50%)
5	4009 (NI)	1002 (50%)

• NI - Growth was not inhibited.

CONCLUSIONS

The present paper describes the synthesis of five new eremantholide C derivatives (2-6) that were spectroscopically characterized and had their activity evaluated in vitro against trypomastigotes of Y and CL strains of T. cruzi. All the eremantholide C derivatives tested (2, 4 and 5) showed 50% growth inhibition of the CL strain.

ACKNOWLEDGMENTS

The authors acknowledge Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support and fellowships.

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Publication Dates

• Publication in this collection
  Dec 2014

History

• Received
  4 Apr 2014
• Accepted
  8 July 2014